inhibiting infections by xcm - cotton incorporated€¦ · inhibiting infections by xcm ....

Libo Shan

Department of Plant Pathology & Microbiology Institute for Plant Genomics & Biotechnology

Texas A&M University

Inhibiting infections by Xcm

Cotton Inc. April 2016


Inhibiting infections by Xcm

Understand biological & genetic

basis of Xcm infections in cotton

Develop biological means to control or prevent

Xcm infections

Cotton Research

Long growth cycle Difficult Complex

recalcitrant for transformation

Cotton

Soft Cool Stylish Versatile Environmentally friendly Natural Fashionable Comfortable Quality fabric

Youthful Traditional Breathes Sporty


natural textile fiber & major oilseed crop 1st value-added crop in USA Unique model for cell wall biosynthesis &

polyploidization Diversity of germplasm 45 diploid species (A or D genome) 5 tetraploid species (AD genome) Complexity of genome G. hirsutum : ~2.5 Gb (95% cotton production)

Importance & complexity of the cotton genome

Gossypium spp.

Li et al., Nat Genet 2014 Wang et al., Nat Genet 2012 Paterson et al., Nature 2012

Li et al., Nat Biotechnol 2015 Zhang et al., Nat Biotechnol 2015


2012: G. raimondii ~41,000 protein-coding genes

2014: G. arboreum ~41,000 protein-coding genes

2015: G. hirsutum ~32,000 A-subgenome ~34,000 D-subgenome

So many genes in cotton!


How to study cotton gene functions? Functional Genomics

Explore cotton gene functions in cotton NOT other plants.

Transient loss-of-function assays (two

weeks) Agrobacterium-mediated VIGS

Transient gain-of-function assays (two

days) Agrobacterium-mediated transient assay Cotton protoplast transfection assay


Modified from Becker and Lange, Trends in Plant Science. 2010

Virus-induced gene silencing

RISC

wheat

maize

barley

grape

pepper

soybean

cabbage

Arabidopsis

tobacco

tomato

rice


Agrobacterium-mediated VIGS

10-day-old seedling

Agro-infiltration

Gao et al., JoVE. 2011

Phenotypic analysis 2-week-post-infiltration

VIGS-vector VIGS-CLA1

Gao et al., The Plant Journal. 2011

CLA1: Cloroplastos alterados 1 involved in chloroplast development


Vector GhCLA1

Deltapine 90

AtCLA1

R135

Phytogen 480WR

Phytogen 425RF

VIGS in diverse cotton genetic backgrounds

Gao et al., The Plant Journal. 2011 Cotton Inc. April 2016

Cotton Functional Genomics

Transient loss-of-function assay

Mining essential cotton genes for the traits

Genetic engineering of the master regulator

Increase sustainable yield, resistance and quality


2X 5X 10X 20X 50X 100X

Cotton functional genomics via VIGS library

~1.9×106 clones G. hirsutum.

10-day-old seedlings

Inoculation with Agro-VIGS cDNA library

Target genes are silenced two weeks after inoculation

Screening for disease resistance or other phenotypes

Identifying interested candidates

Sequencing plasmid DNA from Agrobacteria

Targeted breeding program or transgenic approach


Transient gain-of-function assay in cotton

Plant

Leave

Cell

Signals

Pathogens

Hormones

Abiotic stresses

Light

Transient Gene Function Assays


Subcellular localization in cotton protoplasts


GFP

Plasma membrane-GFP

DIC GFP Hoechst

Nuclear-GFP


GhMAPK activity

GhMPK6 expression

Activation of convergent MAP kinase signaling by pathogen elicitors

Immuno-complex kinase assay to detect cotton MAPK6 activity

Gao et al., The Plant Journal. 2011 Cotton Inc. April 2016

Verticillium wilt Fusarium wilt

RLKs & RLPs

Bacterial blight

TALE targets

Root Rot Drought tolerance

PARP & MAPKs

Cotton comparative, functional and biochemical genomics



Cotton receptor-like protein GhVe1 and receptor-like kinase GhBAK1 mediate

Verticillium wilt resistance.

GhVe1

VIGS-Ctrl GhVe1

GhBAK1

VIGS-Ctrl GhBAK1

VIGS-control

VIGS-GhVe1

VIGS-GhBAK1

Gao et al., Journal of Integrative Biology. 2013 Cotton Inc. April 2016

RLK/RLP complex in cotton Verticillium wilt resistance

Cotton-Verticillium Arabidopsis-Pseudomonas

Felix et al., Plant J. 1999; Gomez-Gomez & Boller, Mol Cell 2000; Chinchilla et al, Plant Cell 2006; Chinchilla et al, Nature 2007; Heese et al., PNAS 2007; Sun et al., Science 2013

AtFLS2

flagellin-triggered immunity

AtBAK1

MAP kinase cascade

Flagellin

GhBAK1

Verticillium resistance

GhVe1

MAP kinase cascade?

Ave1 or ?

GhSoBIR1?



VIGS-Gene 1 VIGS-Control.

Cotton resistance to Fusarium wilt The role of receptor kinases

FOV10 RACE1

H2O

unpublished

RNA-seq to mine novel genes in cotton drought stress

unpublished

RNA-seq: upregulated genes upon 2-day and 4-day drought treatment

ATEXLB1

Control Drought Control Drought

PEBP

SAG12

Gh Actin

ABI 5

MYB43

LRR-RLK

DP491 FM9160 706V

VIGS Ctrl WRKY Myb RLK


Control SnRK MPK CBF3 NFYA5 MYB2 MYC2 ABA8 DREB

Determinatives of cotton drought stress

Two weeks after VIGS; Drought treatment: 12 days

unpublished Cotton Inc. April 2016

Pathogenicity of Xcm The Type III Secretion System

Bacterial blight of cotton (BBC)

Delannoy et al., Annu. Rev. Phytopathol. 2005 Cotton Inc. April 2016

Cotton Inc. April 2016 Yang et al., MPMI 1994

Water-soaking symptom Bacterial release from

mesophyll to leaf surface

Xcm WT

Xcm avrb6 mutant

Xcm avrb6 mutant

Xcm WT

Type III Effectors

Transcription Activator-Like (TAL) Effectors

Moscou and Bogdanove, Science 2009 Mak et al., Science 2012


1 2 3 4 5 6 7 8 9 10 11 12 13 13.5

Avrb6 RVD

EBE

0

HD

N

I

N

G

H

D

H

D

N

I

H

D

N

I

N

S

H

D

H

D

H

D

N

N

N

G

50%

100%

Repeat#

• One of the major virulence factors from XcmH1005 (Race 4).

• Induces strong water-soaking on specific lines of

cotton ~10 TALEs cloned from Xcm, but no any R or S genes have been found from cotton.

TAL effector-host gene recognition

Yang et al., MPMI 1994

Effector Binding Element


Avrb6 Avrb6 PthN PthN Ctrl Ctrl

108 Kd WB: -HA

CBB

Determinatives of TAL effectors in cotton bacterial blight

In collaboration with Dr. Adam Bogdanove


Biological inhibiting infection by Xcm

CRISPR-Cas9 System Revolutionary

genome editing tool

Mutate TALe recognition

site in cotton

Create Cotton that has broad

spectrum resistance to

BBC

Used in rice to create

resistant rice to BBR

Biological inhibiting Xcm infection specifically blocking the effector binding site without interfering its endogenous functions


Acknowledgements

Texas A&M Univ.

Kevin Cox

Dr. Fanhong Meng

Dr. Fangjun Li

Tri Tran

Dr. Xiquan Gao

Dr. Maoying Li

Dr. Ping He

Dr. Tom Isakeit

Texas Agricultural

Experiment Station Lubbock

Dr. Terry Wheeler

USDA-ARS, Texas A&M Univ.

Dr. John Yu

Donald Danforth Plant Science Center Dr. Rebecca Bart Mark Wilson


Cornell Univ. Dr. Adam Bogdanove Katie Wilkins

Boyce Thompson Institute Dr. Zhangjun Fei Dr. Yi Zheng

Univ. of Florida Dr. Dean Gabriel

Texas A&M AgriLife Research Dr. Jane Dever

inhibiting infections by xcm - cotton incorporated€¦ · inhibiting infections by xcm ....

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